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Am J Physiol Regul Integr Comp Physiol 274: R1031-R1038, 1998;
0363-6119/98 $5.00
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Vol. 274, Issue 4, R1031-R1038, April 1998

Functional role of ecto-ATPase activity in goldfish hepatocytes

Pablo J. Schwarzbaum1, Michael E. Frischmann1,2, Gerhard Krumschnabel2, Rolando C. Rossi1, and Wolfgang Wieser2

1 Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, 1113 Buenos Aires, Argentina; and 2 Institut für Zoologie, Abteilung für Ökophysiologie, Universität Innsbruck, A-6020 Innsbruck, Austria

Extracellular [gamma -32P]ATP added to a suspension of goldfish hepatocytes can be hydrolyzed to ADP plus gamma -32Pi due to the presence of an ecto-ATPase located in the plasma membrane. Ecto-ATPase activity was a hyperbolic function of ATP concentration ([ATP]), with apparent maximal activity of 8.3 ± 0.4 nmol Pi · (106 cells)-1 · min-1 and substrate concentration at which a half-maximal hydrolysis rate is obtained of 667 ± 123 µM. Ecto-ATPase activity was inhibited 70% by suramin but was insensitive to inhibitors of transport ATPases. Addition of 5 µM [alpha -32P]ATP to the hepatocyte suspension induced the extracellular release of alpha -32Pi [8.2 pmol · (106 cells)-1 · min-1] and adenosine, suggesting the presence of other ectonucleotidase(s). Exposure of cell suspensions to 5 µM [2,8-3H]ATP resulted in uptake of [2,8-3H]adenosine at 7.9 pmol · (106 cells)-1 · min-1. Addition of low micromolar [ATP] strongly increased cytosolic free Ca2+ (Ca2+i). This effect could be partially mimicked by adenosine 5'-O-(3-thiotriphosphate), a nonhydrolyzable analog of ATP. The blockage of both glycolysis and oxidative phosphorylation led to a sixfold increase of Ca2+i and an 80% decrease of intracellular ATP, but ecto-ATPase activity was insensitive to these metabolic changes. Ecto-ATPase activity represents the first step leading to the complete hydrolysis of extracellular ATP, which allows 1) termination of the action of ATP on specific purinoceptors and 2) the resulting adenosine to be taken up by the cells.

ATP diphosphohydrolase; nucleotidases; adenosine; ATP; metabolic inhibition; fish


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